Phase mask for structured illumination

ABSTRACT

An embodiment of a phase mask is described that comprises a light blocking layer disposed on a substrate, where the light blocking layer has a number of optically transmissive regions each configured as a first pattern. The first pattern includes two segments that have different phase configurations from each other, and the light blocking layer includes at least three angular orientations of the first pattern.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. § 120 ofpending U.S. application Ser. No. 17/176,719, filed Feb. 16, 2021, whichapplication claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 62/978,351, filed Feb. 19, 2019. The entirecontents of the aforementioned applications are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention is generally directed to a phase mask configuredto generate fringe patterns for Structured Illumination Microscopy.

BACKGROUND

It is generally appreciated that Structured Illumination Microscopy(SIM) systems are available commercially on a variety of fluorescencemicroscopes. However, these SIM systems typically use “wide field”illumination. The term, “wide field illumination, as used herein,generally refers to the illumination of a large sample area by sendingcollimated light from a source through a focusing lens before enteringan objective lens element. The normal intent of fluorescence imaging isnot to collect an entire spectrum, but rather to simply filter theemission light for the wavelength of interest and then direct it into acamera. Typically, wide field microscopy is suitable in a variety offluorescence imaging applications, but there are also cases in which aconfocal microscope is superior. Confocal microscopes utilize a pinhole(also sometimes referred to as an aperture) to reject light that is outof focus, thereby vastly improving imaging and is particularly usefulfor imaging through thick samples. Since a confocal image is constructedpixel-by-pixel, rather than over a large area (as in wide fieldimaging), confocal microscopy is well adapted for spectroscopic imagingbecause the emission or scattered light can be sent into a spectrographdownstream of the pinhole. Specific uses for confocal microscopy includefluorescence sectioning through thick, non-homogenous samples, as wellas hyperspectral imaging, such as in the case of imaging Ramanmicroscopy.

An example of an application of SIM in a confocal microscope isdescribed in U.S. application Ser. No. 16/837,512, titled “EnhancedSample Imaging Using Structured Illumination Spectroscopy”, filed Apr.1, 2020, which is hereby incorporated by reference herein in itsentirety for all purposes. For example, the '512 application describesscanning a sample point-by-point, multiple times using fringe patternsthat take advantage of the fact that what is typically referred to as aninterference fringe (e.g. a pattern of evenly spaced alternating brightand dark bands due to light being in our out of phase) can have a finerperiodicity than a focused beam. The '512 application describes usingwhat is referred to as a spatial light modulator (SLM) to generate thefringe patterns.

Those of ordinary skill in the art appreciate that embodiments of SLM'sare well known and are capable of modulating both the intensity andphase of an illumination beam spatially, which is important for combinedconfocal-structured illumination microscopy applications. However, whilean SLM is an excellent device for generating any arbitrary pattern ofintensity and phase, it is generally a poor device to use in acommercialized product. For example, embodiments of SLM are generallyprohibitively expensive, inefficiently utilize light power, and requirea variety of complex optical and electronic overhead.

Therefore, a need exists for a device that is capable of spatiallymodulating the intensity and phase of an illumination beam, and does notsuffer from the drawbacks of an SLM.

SUMMARY

Systems, methods, and products to address these and other needs aredescribed herein with respect to illustrative, non-limiting,implementations. Various alternatives, modifications and equivalents arepossible.

An embodiment of a phase mask is described that comprises a lightblocking layer disposed on a substrate, where the light blocking layerhas a number of optically transmissive regions each configured as afirst pattern. The first pattern includes two segments that havedifferent phase configurations from each other, and the light blockinglayer includes at least three angular orientations of the first pattern.

In some implementations the substrate is constructed of opticallytransparent glass that may include BK7 glass. The optically transparentglass may also include an anti-reflective coating and the light blockinglayer can include a layer of chrome disposed on the substrate. Further,the optically transmissive openings may be radially distributed on thesubstrate in some cases having six instances of the first patterndistributed around with two instances of the first pattern at eachangular orientation.

Also, the two segments may be configured as a circular segment that, insome implementations can have a first side with an arc shape and asecond side that with a substantially linear shape. The three angularorientations of the first pattern may include an angle of 0, pi/3, and2pi/3. In addition, the phase configuration for a first segment maycomprise a phase delay of zero and the phase configuration for a secondsegment may comprise a phase delay of pi, where the second segment mayhave a longer optical path length than the first segment. In someinstances, this is accomplished having a coating of material in thesecond segment.

Further, an embodiment of confocal microscope is described that includesa light source configured to produce a light beam, and a phase mask. Thephase mask has a light blocking layer disposed on a substrate, where thelight blocking layer has with a number of optically transmissive regionseach configured as a first pattern. The first pattern includes twosegments that have different phase configurations from each other andthe light blocking layer includes at least three angular orientations ofthe first pattern. The confocal microscope also includes a deviceoperatively coupled to the phase mask that moves the phase mask toposition the optically transmissive openings in a path of the lightbeam.

In some implementations, the optically transmissive openings include sixinstances of the first pattern, with two instances of the first patternat each angular orientation. In some cases, the three angularorientations include an angle of 0, pi/3, and 2pi/3 and may include aphase configuration for a first segment that comprises a phase delay ofzero and a phase configuration for a second segment that comprises aphase delay of pi. The phase configurations can include a second segmentthat has a longer optical path length than the first segment that insome cases some may be accomplished using a coating of material in thesecond segment.

Additionally, an embodiment of confocal microscope is described thatincludes a light source configured to produce a light beam, a detectorconfigured to produce a signal in response to light from a sample, and aphase mask. The phase mask has a light blocking layer disposed on asubstrate, where the light blocking layer has with a number of opticallytransmissive regions each configured as a first pattern. The firstpattern includes two segments that have different phase configurationsfrom each other and the light blocking layer includes at least threeangular orientations of the first pattern. The confocal microscope alsoincludes a device operatively coupled to the phase mask that moves thephase mask to position the optically transmissive openings in a path ofthe light from the sample.

In some implementations, the light from the sample is produced from aninteraction of the light beam with the sample.

The above embodiments and implementations are not necessarily inclusiveor exclusive of each other and may be combined in any manner that isnon-conflicting and otherwise possible, whether they are presented inassociation with a same, or a different, embodiment or implementation.The description of one embodiment or implementation is not intended tobe limiting with respect to other embodiments and/or implementations.Also, any one or more function, step, operation, or technique describedelsewhere in this specification may, in alternative implementations, becombined with any one or more function, step, operation, or techniquedescribed in the summary. Thus, the above embodiment and implementationsare illustrative rather than limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings. In the drawings, like reference numerals indicatelike structures, elements, or method steps and the leftmost digit of areference numeral indicates the number of the figure in which thereferences element first appears (for example, element 110 appears firstin FIG. 1 ). All of these conventions, however, are intended to betypical or illustrative, rather than limiting.

FIG. 1 is a functional block diagram of one embodiment of a confocalmicroscope in communication with a computer;

FIG. 2 is a simplified graphical representation of one embodiment of theconfocal microscope of FIG. 1 with a phase mask;

FIG. 3 is a simplified graphical representation of a top view of oneembodiment of the phase mask of FIG. 2 illustrating a plurality ofpatterns in a light blocking layer on a substrate;

FIG. 4 is a simplified graphical representation of a side view of oneembodiment of the phase mask of FIG. 3 illustrating a base and fastenerthat holds the substrate;

FIG. 5 is a simplified graphical representation of a comparison of animage collected using the phase mask of FIG. 3 to an image collectedusing a standard confocal arrangement; and

FIG. 6 is a simplified graphical representation of a comparison of animage collected using the phase mask of FIG. 3 to an image collectedusing a standard confocal arrangement.

Like reference numerals refer to corresponding parts throughout theseveral views of the drawings.

DETAILED DESCRIPTION OF EMBODIMENTS

As will be described in greater detail below, embodiments of thedescribed invention include a phase mask configured to spatiallymodulate the intensity and phase of an illumination beam. Morespecifically, the Phase Mask is configured for SIM using a confocalmicroscope enabled for Raman Spectroscopy and/or FluorescenceSpectroscopy.

FIG. 1 provides a simplified illustrative example of user 101 capable ofinteracting with computer 110 and microscope 120. Embodiments ofconfocal microscope 120 may include a variety of commercially availablemicroscopes. For example, confocal microscope 120 may include the DXRconfocal enabled Raman microscopes available from Thermo FisherScientific. FIG. 1 also illustrates a network connection betweencomputer 110 and confocal microscope 120, however it will be appreciatedthat FIG. 1 is intended to be exemplary and additional or fewer networkconnections may be included. Further, the network connection between theelements may include “direct” wired or wireless data transmission (e.g.as represented by the lightning bolt) as well as “indirect”communication via other devices (e.g. switches, routers, controllers,computers, etc.) and therefore the example of FIG. 1 should not beconsidered as limiting.

Computer 110 may include any type of computing platform such as aworkstation, a personal computer, a tablet, a “smart phone”, one or moreservers, compute cluster (local or remote), or any other present orfuture computer or cluster of computers. Computers typically includeknown components such as one or more processors, an operating system,system memory, memory storage devices, input-output controllers,input-output devices, and display devices. It will also be appreciatedthat more than one implementation of computer 110 may be used to carryout various operations in different embodiments, and thus therepresentation of computer 110 in FIG. 1 should not be considered aslimiting.

In some embodiments, computer 110 may employ a computer program productcomprising a computer usable medium having control logic (e.g. computersoftware program, including program code) stored therein. The controllogic, when executed by a processor, causes the processor to performsome or all of the functions described herein. In other embodiments,some functions are implemented primarily in hardware using, for example,a hardware state machine. Implementation of the hardware state machineso as to perform the functions described herein will be apparent tothose skilled in the relevant arts. Also in the same or otherembodiments, computer 110 may employ an internet client that may includespecialized software applications enabled to access remote informationvia a network. A network may include one or more of the many types ofnetworks well known to those of ordinary skill in the art. For example,a network may include a local or wide area network that may employ whatis commonly referred to as a TCP/IP protocol suite to communicate. Anetwork may include a worldwide system of interconnected computernetworks that is commonly referred to as the internet, or could alsoinclude various intranet architectures. Those of ordinary skill in therelated art will also appreciate that some users in networkedenvironments may prefer to employ what are generally referred to as“firewalls” (also sometimes referred to as Packet Filters, or BorderProtection Devices) to control information traffic to and from hardwareand/or software systems. For example, firewalls may comprise hardware orsoftware elements or some combination thereof and are typically designedto enforce security policies put in place by users, such as for instancenetwork administrators, etc.

As described herein, embodiments of the described invention include aphase mask for SIM with a confocal microscope. In the describedembodiments, the phase mask is particularly useful for RamanSpectroscopy and/or Fluorescence Spectroscopy using SIM. For example, asdescribed above the phase mask has a substantially higher level ofefficiency and lower cost than an SLM, as well as being easier toimplement (e.g. the phase mask does not need the additional opticalcomponents and software required by the SLM in order to operateeffectively).

FIG. 2 provides a simplified illustrative example of confocal microscope120 that includes phase mask 200. Confocal microscope 120 includeselements typically found in commercially available confocal microscopessuch as source 215 that produces light beam 217. Source 215 may includeany type of light source used for confocal microscopy that includes, butis not limited to, a laser, Light emitting Diode (LED), broad band, orother type of source known to those of ordinary skill in the art.Embodiments of confocal microscope 120 may also include beam splitter225 that selectively reflects light in a specified wavelength range toobjective lens 227 and sample 205, and is transmissive within aspecified wavelength range to allow light to pass through to lens 229through aperture 223 (e.g. a “pinhole” type aperture) to detector 235.Detector 235 may include any type of detector typically found incommercially available confocal microscopes such as a CCD,photomultiplier, or other type of detector known to those of ordinaryskill in the art. Those of ordinary skill in the related art will alsoappreciate that the FIG. 2 is provided for the purposes of example andthat other elements and/or configurations of confocal microscope 120 areconsidered within the scope of the described invention. For example,prior to reaching detector 235 the light may first pass through aspectrograph to disperse the light into a spectrum.

In the example of FIG. 2 , phase mask 200 is positioned in the path oflight beam 217 to pattern the excitation light delivered to sample 205.However, it will also be appreciated that phase mask 200 can bepositioned in the path of light 219 to pattern the light from sample 205onto detector 235 (e.g. light that is emitted, scattered, etc. as aresult of an interaction of light beam 217 with sample 205).

FIG. 3 provides an illustrative example of a top view of phase mask 200.Embodiments of phase mask 200 include substrate 307 constructed from anoptically transmissive material with known optical characteristics. Forexample, substrate 307 may include a 60 mm×60 mm area constructed from atype of optical glass used in lenses and other optical components, suchwhat is referred to as “crown glass” that has good optical andmechanical characteristics, and is resistant to chemical andenvironmental damage. One particular type of crown glass useful forphase mask 200 includes glass with a borosilicate additive, such as BK7glass available from Schott AG.

As illustrated in FIG. 3 , substrate 307 includes light blocking region305 that may include any type of configuration capable of blocking thetransmission of light through substrate 307. One example includes aconfiguration that comprises a deposition of a layer of chrome materialon the surface of substrate 307 (e.g. may be the top surface or bottomsurface). Also, FIG. 3 illustrates light blocking region 305 as asubstantially circular ring, however it will be appreciated that thering configuration is exemplary and other configurations may be utilized(e.g. a substantial portion the surface area of one side substrate 307may include light blocking region 305, or light blocking region 305 maybe configured as a linear strip).

Further, FIG. 3 illustrates a plurality of optically transmissiveregions in light blocking region 305, illustrated as pattern 310. Asdescribed above, the ring configuration of light blocking region 305 andthe arrangement of each of patterns 310, as illustrated in FIG. 3 , isexemplary and should not be considered as limiting. In some embodiments,pattern 310 may include a linear strip of light blocking region 310 or,as described above, may include a substantial portion the surface areaof the surface of substrate 307 where the arrangement of patterns 310may be substantially linear. For example, in the ring or linearconfiguration, pattern 310 may include a pattern covering region ofabout 4 mm×4 mm. For a linear embodiment this may include a linearconfiguration of light blocking region 305 comprising about 4 mm×24 mm

FIG. 3 also illustrates fastener 320 that may include a nut/boltconfiguration or any other fastener configuration known to those ofordinary skill in the art. FIG. 4 illustrates a side view of phase mask200 that includes substrate 307 having a substantially planarconfiguration and substantially consistent thickness that is held inplace by fastener 320 operatively coupled to base 405. In someembodiments, base 405 utilizes clamping mechanism to operatively connectphase mask 200 to a translation device such as a motor that rotatesphase mask 200 about the axis around fastener 320 (e.g. a steppermotor). In embodiments where phase mask 200 includes a lineararrangement of patterns 310, the translation device is constructed andarranged to provide linear motion to phase mask 200. It will also beappreciated that the translation device may include other types ofelements known in the related art, such as a piezo, etc.

In addition, FIG. 3 illustrates 6 instances of pattern 310 at variousangles relative to the optical path of light beam 217 (e.g. when theinstance of pattern 310 is positioned in the optical path). Eachinstance of pattern 310 is at a location of light blocking region 305indicated by position indicator 303. Light blocking region 305 alsoincludes pattern 313 at a first position indicated by position indicator303 that is substantially circular to permit substantially all of lightbeam 217 to pass through substrate 307, and a region without a lighttransmission pattern at position 2 indicated by position indicator 303that blocks substantially all of light beam 217 from passing throughsubstrate 307.

In some embodiments, phase mask 200 includes 3 angular orientations,where there are 2 instances of pattern 310 per angle one instance ofpattern 310 that includes deposited layer segment 315 and substratesegment 317, as well as a second instance of pattern 310 that has twooccurrences of substrate segment 317. Also, pattern 310 may include twosegments arranged as “slit” shaped elements (e.g. a slit includes a longnarrow opening), which may sometimes be referred to as “circularsegments”. Further, in some embodiments the diameter of the circularsegments of pattern 310 are matched to the back-aperture diameter ofobjective lens 227. For example, for an Olympus 100×0.9 NA, thatdiameter would be >=3.24 mm, and for a long working distance Olympus100×0.8 NA, that diameter would be >=2.88 mm. Also in the presentlydescribed example, one side of each circular segment may have an arcshape such as, for instance, a substantially circular shape (e.g. about¼ of a circle), and a second side that is substantially linear.

Importantly, in some instances of pattern 310 there is an optical pathlength difference between the two segments of pattern 310. In otherwords, the segments have a different optical path length from each otherthat creates a phase difference in light passing through (e.g. thesegment with a longer optical path length produces a phase delay in theportion of light beam 217 that passes through it relative to the segmentwith the shorter optical path length). In some embodiments, the opticalpath difference may be created using a deposition of additional materialonto substrate 307 (e.g. substrate 307 comprises two substantiallyplanar surfaces with consistent thickness) in one of the segments ofpattern 310 to produce deposited layer segment 315 that combined withsubstrate 307 comprises the longer optical path when compared tosubstrate segment 317 of pattern 310 that only includes substrate 307.The deposited material may be the same material as substrate 307 orother suitable material. Alternatively, or in combination with thedeposition, removal of material from substrate 307 in a segment may beused to shorten to optical path length of one of the segment of pattern310.

For example, the refractive index difference between BK7 glass used forsubstrate 307 and air creates an optical path difference between thesegments of pattern 310. Since the refractive index of BK7 glass is 1.52at an excitation wavelength of 532 nm, light travels more slowly throughBK7 than it does in air. Therefore, an optical path difference can begenerated through controlled deposition of BK7 onto one of the segments.Those of ordinary skill in the art appreciate that where n is refractiveindex, and d is length, and if d1=d2 (e.g. the light travels the samedistance in air as it does while it is traveling through the BK7), thenthe Optical Path Difference (OPD) is equal to n1*d-n2*d (may also beexpressed as d=OPD/(n1-n2)). In the present example, n1 is 1.0003 forair and n2 is 1.52 for BK7 at an excitation wavelength of 532 nm (e.g.the values are wavelength dependent). For SIM applications a pi phasedifference between the segments is highly desirable, which equates to anOPD of 266 nm for an excitation wavelength of 532 nm. Solving for d, adeposition of BK7 material at a thickness of 512 nm onto one of thesegments produces the desired pi phase difference. Therefore, whenportions of light beam 217 passes through substrate segment 317 anddeposited layer segment 315 that includes a coating of 512 nm of BK7glass, then the phase difference between the light traveling throughsegments 315 and 317 will be pi.

In the embodiment illustrated in FIG. 3 , the 6 instances of pattern 310include 3 different angles of 0, pi/3, and 2pi/3, where for each angle,pattern 310 includes substrate segment 317 with phase delay of 0 anddeposited layer segment 315 with a phase delay of pi. In the describedembodiments, the 6 instances of pattern 310 are useful for performingstructured illumination by allowing light beam 217 to successively passthrough each instance of pattern 310 as it is positioned in the opticalpath of light beam 217 (e.g. rotated into the optical path in the caseof a circular arrangement as illustrated in FIG. 3 , or linearlytranslated for a linear arrangement). For example, embodiments ofconfocal microscope 120 equipped with phase mask 200 and computer 110 toprocess the images, can implement SIM to image a sample and obtain aspatial resolution of 150 nm, that is a 2×improvement over thediffraction limit of 300 nm of a typical confocal microscope.

FIG. 5 provides an illustrative example of a comparison of SIM-Ramanimage 510 and associated SIM-Raman data 515 collected using phase mask200 and confocal image 520 and associated confocal data 525 collectedusing a standard confocal microscopy configuration, where both SIM-Ramanimage 510 and confocal image 520 have the same field of view of asubstrate comprising an arrangement of vertical and horizontal linesseparated by a 250 nm pitch. The example of FIG. 5 clearly demonstratesthat upon visual inspection SIM-Raman image 510 has superior resolutionto confocal image 520, which is further reinforced by SIM-Raman data 515that illustrates superior intensity discrimination for data collectedalong data line 505 over confocal data 525.

FIG. 6 provides a further illustrative example of a comparison ofSIM-Raman image 610 and associated SIM-Raman data 615 collected usingphase mask 200 and confocal image 620 and associated confocal data 625collected using a standard confocal microscopy configuration, where bothSIM-Raman image 610 and confocal image 620 have the same field of viewof a substrate comprising an arrangement of overlapping carbonnanotubes. Again, the example of FIG. 6 clearly demonstrates that uponvisual inspection SIM-Raman image 610 has superior resolution toconfocal image 620, which is further reinforced by SIM-Raman data 615that illustrates superior intensity discrimination for data collectedalong data line 605 over confocal data 625 (e.g. SIM-Raman image 610clearly resolves two separate carbon nanotubes, whereas confocal image620 blurs the image of the two together except for the distal ends ofthe nanotubes).

Having described various embodiments and implementations, it should beapparent to those skilled in the relevant art that the foregoing isillustrative only and not limiting, having been presented by way ofexample only. Many other schemes for distributing functions among thevarious functional elements of the illustrated embodiments are possible.The functions of any element may be carried out in various ways inalternative embodiments

What is claimed is:
 1. A phase mask, comprising: a substrate; and a light blocking layer disposed on the substrate with a plurality of optically transmissive regions each configured as a first pattern that includes two segments having different phase configurations, wherein the light blocking layer comprises three angular orientations of the first pattern, wherein the phase configuration for a first segment comprises a phase delay of zero and the phase configuration for a second segment comprises a phase delay of pi.
 2. The phase mask of claim 1, wherein: the substrate comprises optically transparent glass.
 3. The phase mask of claim 2, wherein: the optically transparent glass comprises BK7 glass.
 4. The phase mask of claim 2, wherein: the optically transparent glass comprises an anti-reflective coating.
 5. The phase mask of claim 1, wherein: the light blocking layer comprises a layer of chrome disposed on the substrate.
 6. The phase mask of claim 1, wherein the plurality of optically transmissive regions are radially distributed on the substrate.
 7. The phase mask of claim 1, wherein: the plurality of optically transmissive openings includes six instances of the first pattern.
 8. The phase mask of claim 7, wherein: the six instances comprise two instances of the first pattern at each angular orientation.
 9. The phase mask of claim 1, wherein: each of the two segments comprise a circular segment comprising a first side with an arc shape and a second side that comprises a substantially linear shape.
 10. The phase mask of claim 1, wherein: the three angular orientations include an angle of 0, pi/3, and 2pi/3.
 11. The phase mask of claim 1, wherein: the second segment comprises a longer optical path length than the first segment.
 12. The phase mask of claim 11, wherein: the second segment comprises a coating of material.
 13. A microscope, comprising: a light source configured to produce a light beam; a phase mask that comprises: a substrate; and a light blocking layer disposed on the substrate with a plurality of optically transmissive regions each configured as a first pattern that includes two segments having different phase configurations, wherein the light blocking layer comprises three angular orientations of the first pattern relative to the light beam; and a device operatively coupled to the phase mask and configured to move the phase mask to position the optically transmissive openings in a path of the light beam.
 14. The microscope of claim 13, wherein: the plurality of optically transmissive openings includes six instances of the first pattern.
 15. The microscope of claim 14, wherein: the six instances comprise two instances of the first pattern at each angular orientation.
 16. The microscope of claim 13, wherein: the three angular orientations include an angle of 0, pi/3, and 2pi/3.
 17. The microscope of claim 13, wherein: the phase configuration for a first segment comprises a phase delay of zero and the phase configuration for a second segment comprises a phase delay of pi.
 18. The microscope of claim 17, wherein: the second segment comprises a longer optical path length than the first segment.
 19. The microscope of claim 18, wherein: the second segment comprises a coating of material.
 20. The microscope of claim 13, further comprising: a detector configured to produce a signal in response to an interaction of the light beam with a sample. 